Field of the Invention
This invention relates generally to the field of insect inhibitory Bacillus thuringiensis proteins and, more particularly, to B. thuringiensis crystal proteins that inhibit hemipteran insects. Isolated polynucleotides and proteins, transgenic plants and related methods that provide for inhibition of hemipteran insects are described. Also described are methods for combining the B. thuringiensis crystal proteins that inhibit hemipteran insects with distinct insect control agents to obtain increased levels of hemipteran insect inhibition, hemipteran insect resistance management, or an expanded spectrum of insect pest control.
Related Art
Bacillus thuringiensis Crystal Proteins
The Gram-positive soil bacterium Bacillus thuringiensis is well known for its production of proteinaceous parasporal crystals, or δ-endotoxins, that are toxic to a variety of Lepidopteran, Coleopteran, and Dipteran larvae. B. thuringiensis produces crystal proteins during sporulation which are specifically toxic to certain species of insects. Many different strains of B. thuringiensis have been shown to produce insecticidal crystal proteins, and compositions comprising B. thuringiensis strains which produce proteins having insecticidal activity have been used commercially as environmentally-acceptable insecticides because of their toxicity to the specific target insect, and non-toxicity to plants and other non-targeted organisms.
Commercial formulations of naturally occurring B. thuringiensis isolates have long been used for the biological control of agricultural insect pests. In commercial production, the spores and crystals obtained from the fermentation process are concentrated and formulated for foliar application according to conventional agricultural practices.
Nomenclature of Crystal Proteins
A review by Hofte et al., (Hofte and Whiteley, Microbiol. Rev., 53:242-255, 1989) describes the general state of the art with respect to the majority of insecticidal B. thuringiensis strains that have been identified which are active against insects of the Order Lepidoptera, i.e., caterpillar insects. This treatise also describes B. thuringiensis strains having insecticidal activity against insects of the Orders Diptera (i.e., flies and mosquitoes) and Coleoptera (i.e., beetles). A number of genes encoding crystal proteins have been cloned from several strains of B. thuringiensis. Hofte et al. (1989) discusses the genes and proteins that were identified in B. thuringiensis prior to 1990, and sets forth the nomenclature and classification scheme which has traditionally been applied to B. thuringiensis genes and proteins. Cry1 genes encode Lepidopteran-toxic Cry1 proteins. Cry2 genes encode Cry2 proteins that are toxic to both Lepidopterans and Dipterans. Cry3 genes encode Coleopteran-toxic Cry3 proteins, while Cry4 genes encode Dipteran-toxic Cry4 proteins, etc.
Recently a new nomenclature has been proposed which systematically classifies the Cry proteins based upon amino acid sequence homology rather than upon insect target specificities. This classification scheme and a comprehensive list of insect inhibitory B. thuringiensis genes is summarized in the listing of Insecticidal Toxin Proteins as set forth in the Neil Crickmore website accessed through Cambridge University on the world wide web at lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/index.html.
Mode of Crystal Protein Toxicity
All δ-endotoxin crystals are toxic to insect larvae by ingestion. Solubilization of the crystal in the midgut of the insect releases the protoxin form of the δ.-endotoxin which, in most instances, is subsequently processed to an active toxin by midgut protease. The activated toxins recognize and bind to the brush-border of the insect midgut epithelium through receptor proteins. Several putative crystal protein receptors have been isolated from certain insect larvae (Jurat-Fuentes J L, Adang M J. Biochemistry. 45(32):9688, 2006; Griffitts J S et al., Science. 307(5711):922, 2005; Jurat-Fuentes J L, Adang M J. Eur J Biochem.; 271(15):3127, 2004). The binding of active toxins is followed by intercalation and aggregation of toxin molecules to form pores within the midgut epithelium. This process leads to osmotic imbalance, swelling, lysis of the cells lining the midgut epithelium, and eventual larvae mortality.
With the advent of molecular genetic techniques, various δ.-endotoxin genes have been isolated and their DNA sequences determined. These genes have been used to construct certain genetically engineered B. thuringiensis products that have been approved for commercial use. Recent developments have seen new δ.-endotoxin delivery systems developed, including plants that contain and express genetically engineered δ.-endotoxin genes. Control of Lepidopteran and Coleopteran pests in a variety of transgenic crop plants including corn, cotton, potato, tomato and rice that express δ.-endotoxin genes is well established. Advantages associated with expression of the δ.-endotoxin genes in crop plants include increased yields and decreased use of chemical insecticides. The advantages of transgenic crops that express insect inhibitory δ.-endotoxin genes has lead to widespread use in crops such as corn and cotton.
Unfortunately, the δ.-endotoxin genes that are currently available do not provide for control of all insect pests that plague crop production. In particular, Hemipteran insects still must be controlled by use of insecticides in crops where they cause damage. The Hemipteran or “piercing/sucking” insects are especially damaging to plants in that they are also known to transmit damaging plant viruses and cause plants to be more susceptible to bacterial and fungal infection. There is thus a need for additional materials and methods that would permit inhibition of Hemipteran insect pests in crops. There is also a need to obtain several different types of Hemipteran insect control agents with distinct modes of action for use in transgenic plants as Hemipteran insect resistance management tools.
Given the need for Hemipteran insect control agents, a variety of approaches have been disclosed. U.S. Pat. No. 5,723,440 describes a Cyt1Bal protein with purported activity against Hemipteran insects. However, Wellman-Desbiens and Cote (J. Econ. Entomol. 98(5):1469-1479., 2005) were unable to confirm this activity with the Hemipteran insect Lygus hesperus. U.S. Pat. No. 5,885,963 discloses the use of B. thuringiensis israelensis Cyt toxins that purportedly inhibit Hemipteran pests. More recently, US20060242732 discloses B. thuringiensis crystal proteins with activity against the Hemipteran insect Lygus lineolaris. These proteins are unrelated to the Cyt proteins described in U.S. Pat. Nos. 5,723,440 and 5,885,963.